Spatiotemporal heterologous vaccination relies on type I interferons at priming to enhance mucosal immunity.

Parenteral vaccines against respiratory viruses poorly block infection and transmission due to suboptimal induction of mucosal immunity. In contrast, hybrid immunity and heterologous-route immunization regimens generate protective mucosal and systemic immunity. Herein, we examine how chronological order in heterologous prime/boost immunization regimens impacts immune responses using combinations of intramuscular mRNA and intranasal adjuvanted subunit vaccines against SARS-CoV-2. Enhanced long-lived mucosal and systemic immunity depended strongly on vaccine delivery sequence, with heterologous regimens administered in alternate order of routes yielding markedly distinct immune profiles with different Th-biases and divergent localizations. Immunity imparted through heterologous immunization was more durable and responsive to delayed boosting than parenteral-only regimens. Furthermore, intramuscular mRNA immunization uniquely shaped gene expression locally and systemically through IFN-I induction, allowing for enhanced responses to subsequent mucosal immunizations. These findings demonstrate that the context of initial antigen exposure-including the route of exposure and associated inflammatory signals-profoundly shapes responses to subsequent encounters. Thus, vaccine responses in antigen-experienced hosts differ fundamentally from those induced on a naive baseline, reflecting the influence of pre-existing innate and adaptive immune memory.